The aims of this research project are to understand the regulation of viral gene expression during herpesvirus infection, evaluate the feasibility of herpesvirus vectors as gene therapy vectors, and determine the herpesvirus genes that would be most effective for development as potential subunit vaccines. Understanding the regulation of HSV gene expression is essential for the development and evaluation of successful vaccines, particularly live virus vaccines. During herpes simplex virus infection, approximately 75 genes are expressed in a well ordered process that can be conveniently divided into three phases based on the temporal order of mRNA synthesis. Immediate-early genes are expressed soon after virus infection, and their gene products are required for the expression of early genes, many of whose gene products are involved in viral DNA replication. Expression of viral late genes requires viral DNA replication and functional immediate-early gene products. In FY00, replication-incompetent herpes simplex viruses have been constructed to express foreign genes only when induced by the antibiotic tetracycline. Several novel vectors containing tet-inducible elements linked to HSV-1 immediate-early promoter have been evaluated for their ability to express foreign genes. These studies have shown that gene expression from such vectors can be regulated and can persist for extended periods of time in non-dividing cells. Work is underway to develop vectors which can supply the tetracycline transactivator that is necessary for induction. Another part of this research project is designed to evaluate various HSV gene products as potential components of subunit vaccines, and to evaluate different delivery strategies in order to maximize a protective immune response. Multiple HSV-2 genes, including all of the open reading frames from the unique short region, have now been cloned into plasmid expression vectors. Gene gun delivery is being used to immunize mice with these constructs before lethal viral challenge. In addition, membrane bound and secreted forms of viral glycoproteins are being compared for their relative ability to stimulate protective immunity. And finally, prime boost strategies that include plasmid expression vectors and vaccinia vectors are being evaluated for their ability to elicit a protective response.